Treatments for immune systems disorders

ABSTRACT

This invention provides methods and formulations for treating disorders of the immune system comprising administering a fortified formulation comprising stabilized rice bran derivative and a fortification agent. Preferred rice bran derivatives are rice bran oil and the solubilized fraction of rice bran. Preferred fortification agents are glucosamine derivative, methylsulfonylmethane, yucca concentrate, and grape seed extract.

This application claims priority to U.S. Application Ser. No.60/307,588, filed Jul. 23, 2001, the disclosure of which is incorporatedherein by reference in its entirety for all purposes.

FIELD OF THE INVENTION

This invention relates to fortified formulations and methods for usingsuch formulations for treating joint inflammation, pain, and loss ofmobility.

BACKGROUND OF THE INVENTION

Joint disorders and injuries are widespread, can cause considerablediscomfort, and cost billions of dollars in lost days of work. Symptomsof these diseases and injuries include inflammation, lameness, loss ofmobility, and pain.

Arthritis is a multifactorial degenerative joint disease, whichprogresses with age and results in joint stiffness, inflammation, andpain. A joint is formed where two bones meet. The healthy joint bonesare lined with spongy cartilage which act as shock absorbers, and thesynovial fluid, which is secreted by the synovial membrane lining thejoint space, acts as a lubricant that prevents friction. There are twomajor types of arthritis, osteoarthritis, and rheumatoid arthritis.

Osteoarthritis is a condition occurring due to the progressivedegeneration and the wearing away of the cartilage (the cushion betweenthe joints), especially at the large joints like the hips and knees. Itis a normal age-related degenerative process, occurring gradually afternormal wear and tear. Being overweight, inheriting the wrong genes, orsimply growing old can make the problem worse. Almost 80% of the peopleover the age of 60 years, all over the world, suffer from this disorder.OA begins with the breakdown of cartilage resulting in pain,inflammation, and progressive stiffness in the joint. Jointstrengthening through exercise, weight maintenance, and non-steroidalanti-inflammatory drugs (NSAIDs) can alleviate symptoms. Osteoarthritisis often accompanied by osteoporosis, a condition where the bone calciumresorption occurs due to hormonal imbalance, making the bone morebrittle, which may lead to frequent fractures.

Rheumatoid arthritis is a prostaglandin-mediated joint disease thatleads to irreversible crippling of small joints, especially the fingersand toes. This condition is difficult to handle and only prostaglandinsynthetase inhibitors can give some relief to patients. Essentially, thebody's immune system attacks the cartilage, and the white blood cells(leukocytes) attack the collagen. Statin drugs, which areimmunomodulators, are used frequently, though they have serious sideeffects. Temporary relief may be obtained from NSAIDs. However, overuseof these drugs can lead to ulcers.

Soft tissue rheumatism is a condition where many parts of the body canbe affected for a variety of reasons. In many instances, soft tissuerheumatism is a sport-induced injury, such as a sprain, tennis elbow, orrunner's knee. Soft tissues are the ligaments, tendons, and tendonsheath. Ligaments are bands of tissue that connect bones. Tendons arebands of tissue that connect muscle to bone. The tendon sheath is thetissue that surrounds and lubricates the tendon. Injury to any of thesesoft tissues can produce inflammation, pain, and stiffness. Theseconditions typically clear up quickly, within several days to weeks, andare usually treated with NSAIDs, icing the affected area, and rest.Nevertheless, expedited recovery is still desirable, as these injuriescan cause considerable discomfort and reduce workplace productivity.

In addition to the use of NSAIDs, the inflammatory response can beregulated through the use multiple other drugs (see, Goodman & Gilman's“The Pharmacological Basis of Therapeutics” eds. Hardman et al. NinthEdition, McGraw-Hill Publishing, 1996). Unfortunately, certainanti-inflammatory drugs presently available produce cytotoxic effectsthat reflect their initial employment as cancer chemotherapeutics,typically anti-neoplastics. For example, corticosteroids, which areoften used for treatment of acute inflammation, manifest significantadverse effects, such as inducing Cushingoid features, skin thinning,increased susceptibility to infection, and suppression of thehypothalamic-pituitary-adrenal axis.

Joint injuries and conditions as described above also afflict numerousother mammals, including domesticated animals such as dogs, cats, andhorses. In particular, horses often sustain considerable joint injuriesdue to their participation in sporting events or use for farm work.Lameness due to traumatic joint disease is a common clinical problem inhorses and is one of the most important sources of financial losses inthe equine industry.

In recent years NSAIDS (non-steroidal anti-inflammatory drugs), such asphenylbutazone, have been used to eliminate, diminish, or at leastassist in managing the lameness in performance horses in all aspects ofthe horse industry (including racing, cutting, reigning, hunter-jumper,dressage, rodeo, barrel-racing). Unfortunately, NSAIDS requireprescriptions and/or veterinary dispensing, are costly, and areaccompanied by mild severe, and sometimes even catastrophic sideeffects.

As described previously, such methods for treatment of both humans andanimals only allow temporary relief and/or exhibit side effects fromprolonged use. Therefore, there is a need for safe and effectivetreatment which can be used on a long-term basis without side effectsand which also promotes rebuilding of the injured/diseased joints. Thepresent invention fulfills this and other needs.

BRIEF SUMMARY OF THE INVENTION

It has now been surprisingly found that a stabilized rice branderivative, having significant amounts of potent phytonutrients andantioxidants, has an excellent effect in relieving arthritic pain andpain associated with inflammation. Fortification of these derivativeswith certain minor herbal components enhances their action. Thefortified formulations comprising a stabilized rice bran derivative anda fortification agent reduce inflammation, pain, lameness, and loss ofmobility. These fortified formulations are more effective, have moreimmediate action, and require lower dosages than currently existingformulations for such conditions.

As such, the present invention provides a method for treating aninflammatory disease or reducing an inflammatory reaction in a mammalcomprising administering a stabilized rice bran derivative and afortification agent. The stabilized rice bran derivative can include,but is not limited to rice bran oil, enzyme-treated stabilized ricebran, a solubilized fraction, or mixtures thereof. Preferably, thestabilized rice bran derivative is rice bran oil or the solubilizedfraction. The fortification agent can include, but is not limited to, aglucosamine derivative, methylsulfonylmethane, yucca concentrate, orgrape seed extract. In preferred embodiments, the administeringcomprises ingestion of the formulation and the inflammatory disease is adisorder of the bone joint, including, but not limited to,osteoarthritis, osteoporosis, rheumatoid arthritis, and soft tissuerheumatism. The mammal is typically a human or a horse.

In another aspect, the present invention provides a method for treatinglameness or loss of mobility in a mammal comprising administration of aformulation comprising a stabilized rice bran derivative and afortification agent.

In yet another aspect, the present invention provides a method forreducing pain in a mammal, the method comprising administration of aformulation comprising a stabilized rice bran derivative and afortification agent.

In still yet another aspect, the present invention provides a method forreducing prostaglandin synthetase activity, the method comprisingadministration of a formulation comprising a stabilized rice branderivative and a fortification agent.

In another aspect, the invention provides a formulation comprising astabilized rice bran derivative and fortification agent for treatingjoint inflammation and loss of mobility. In certain embodiments, thefortification agent is a glucosamine derivative, methylsulfonylmethane,yucca concentrate, or grape seed extract.

Also provided are formulations and methods for treatment of aninflammatory disease, lameness, and loss of mobility comprisingadministering a tocol composition.

These and other aspects will become more apparent when read with thedetailed description and examples, which follow.

DEFINITIONS

Unless otherwise specified, the following terms used in thespecification and claims have the meanings given below.

The term “fortification agent” refers to any agent which improves theability of the stabilized rice bran derivative to treat a pathologicalcondition of the joint, such as an inflammatory disease, lameness, lossof mobility, pain. etc. Preferred fortification agents includeglucosamine derivatives, methylsulfonylmethane, yucca concentrate, grapeseed extract and combinations thereof.

The term “glucosamine derivative” refers to modified versions ofglucosamine, such as glucosamine sulfate and N-acetyl glucosamine. Thesederivatives are the building blocks of proteoglycans, which are used incartilage synthesis.

The term “methylsulfonylmethane” (MSM) refers to a chemical compoundthat is a sulfur donor. Depletion of sulfur amino acids leads toarthritis. Sulfur compounds aid in the synthesis of proteoglycans andglucosaminoglycans, which form the basic matrix of cartilage.

The term “yucca concentrate” refers to a preparation of the root of theyucca plant. Yucca is a desert plant that is rich in a steroidal saponin(sarasaponin). This saponin helps the body's production of cortisone andimproves the ratio of cortisol/DHEA.

The term “grape seed extract” refers to an extract from grape seedswhich has antioxidant effects.

The term “administering” refers to the manner in which a therapeuticagent is introduced to a mammal with a particular condition. Suchadministration may be by any one of the various standard routes foradministration of drugs, i.e., topical, oral (ingestion by the mammal),parenteral, etc.

The term “reducing lameness or loss of mobility” refers to improving thefunction of any joint. Joint function can be measured by evaluatingparameters such as the range of motion and presence of discomfort orpain during movement.

The term “reducing pain in or around a bone joint” refers to alleviatingpain localized around a bone joint.

The term “prostaglandin synthetase” (also termed prostaglandinendoperoxide synthase) refers to the cyclooxygenase (COX) enzymes whichcatalyze the conversion of arachidonic acid to prostanoids.

The term “disorder of the bone joint” refers to any condition where thenormal function of the joint, i.e., range of motion, ability to bearweight and the like is impaired, or where use of the joint isaccompanied by discomfort or pain. These conditions include those whereimpairment of the joint is the primary symptom or ones where thedisorder has multiple symptoms. These disorders typically affect thebone, joint capsule, cartilage, tendons, ligaments, tendon sheaths,bursa, synovial fluid, etc. Common disorders of the joint includeosteoarthritis, rheumatoid arthritis, gout, lupus, tendonitis, bursitis,carpal tunnel syndrome, sprains, etc.

The term “osteoarthritis” refers to a degenerative joint disease wherethe cartilage that normally cushions the joint and protects it fromimpact erodes.

The term “rheumatoid arthritis” refers to an autoimmune disease withinflammation of the lining of the joints (synovial membrane). Thethickened synovial membrane can erode the surrounding ligaments andbone.

The term “soft tissue rheumatism” refers to conditions where the softtissues of the body, such as ligaments, tendons, and the tendon sheathare injured.

The term “tocol” refers to E complex vitamins known as tocopherols andtocotrienols which have antioxidant properties. There are at least tendifferent isomeric forms of these vitamins. The term “tocol composition”refers to any composition comprising tocols.

As used herein the term “enzyme treated stabilized rice bran derivative”refers to an enzyme-treated stabilized rice bran made by mixing astabilized rice bran with an aqueous solution in a 15% to about a 35%aqueous slurry w/w; adding an enzyme to the aqueous rice bran slurry toconvert starch to dextrin, and then directly drying the dextrin solutionto form an enzyme treated stabilized rice bran derivative. The enzymetreated stabilized rice bran comprises about 20% to about 30% totaldietary fiber.

As used herein the term “GRAS” means generally regarded as safe withrespect to food additives.

As used herein the term “stabilized rice bran derivative solubilizedfraction” refers to a fraction during a partitioning process.Specifically, after the stabilized rice bran aqueous slurry isenzymatically treated, it is then pumped into a centrifuge where theinsoluble fraction precipitates out of the aqueous solution. The aqueousmaterial is pumped to a dryer and then dried. This dried aqueous portionproduces the soluble fraction. The constituent parts and theirpercentages are listed in the Tables below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the standards used to score the degree of lameness ofhorses. Horses are evaluated for extension, toe placement, and flexion.

FIG. 2 illustrates the effect of formulations used in methods of thisinvention versus other commercially available treatments/NSAIDs onlameness of horses. The graph measures soundness score (measurement oflameness) after treatment with “bute”, Cosequin, Absorbine Flex over 50days.

FIG. 3 illustrates the effect of treatment with formulations of thisinvention versus other commercially available nutraceuticals/NSAIDs onlevels of carrageenan-induced inflammation on Day 21 of the clinicaltrial. Injection is induced and then the size of inflammation at thesite of injection with carrageenan is measured over 7 hours for horsestreated with the negative control, “bute”, Cosequin, Next Level, orAbsorbine Flex.

FIG. 4 illustrates the effect of treatment with formulations of thisinvention versus other commercially available nutraceuticals/NSAIDs onlevels of carrageenan-induced inflammation on Day 50 of the clinicaltrial.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS I.COMPOUNDS USED IN METHODS AND FORMULATIONS OF THIS INVENTION

Compounds used in formulations and methods of this invention includestabilized rice bran derivatives, which can include, but are not limitedto rice bran oil, enzyme-treated stabilized rice bran, a solubilizedfraction, or mixtures thereof. Preferably, the stabilized rice branderivative is rice bran oil or the solubilized fraction. Other compoundsused in formulations and methods of this invention include fortificationagents which can include, but are not limited to, a glucosaminederivative, methylsulfonylmethane, yucca concentrate, grape seedextract, curcumin, ginger powder, boswellin, aswagandha, and hempseedoil. The compounds of the invention may also comprise an extract ofactive ingredients of rice bran derivatives, such as tocols.

A. Stabilized Rice Bran Derivatives

In harvested rice, also known as rough rice, the kernel is completelyenveloped by the rice hull. The milling process removes the hull, whichyields brown rice. The outer brown layer is then removed by an abrasivemilling process to generate white rice. The separated brown layer isdesignated rice bran.

Rice bran is the mesocarp, i.e., the portion between the hull and ricegrain, obtained by milling or polishing brown rice. It constitutes about10% of rough rice. It is generally used as an animal feed. It containsabout 18-24% fat, about 25% dietary fiber, about 14% protein and about45% total carbohydrates besides several potent micronutrients. It isrich in B-complex vitamins, vitamin E and its isomers, minerals likepotassium, magnesium, and phosphorous besides several potentantioxidants.

Stabilized rice bran can be commercially purchased or prepared usingvarious methods. Most stabilization methods of rice bran result ininactivation of the lipases which are present, inactivation of theperoxidases, and inactivation of the microorganisms, while stillmaintaining the high levels of antioxidants in the rice bran. For ageneral discussion of stabilization and processing see, Rice Science andTechnology, edited by W. E. Marshall and James I Wadswoth, (1994) pages390-404.

Stabilized rice bran is available commercially from Producers Rice MillInc. (Stuttgart, Ark.), Riceland Foods (Stuttgart, Ark.), Riviana Foods,Inc. (Houston, Tex.), Uncle Ben's Inc. (Houston, Tex.) and The RiceX™Company (El Dorado Hills, Calif.). Due to different stabilizationprocesses, stabilized rice bran will differ in composition andstabilization characteristics when derived from different manufacturers.

In order to generate the rice bran derivatives for use in the presentinvention, the rice bran is first stabilized, and then it is furtherseparated into at least two fractions. These include, but are notlimited to, a stabilized rice bran soluble derivative and a stabilizedrice bran insoluble derivative. Preferably, the separation into the ricebran derivatives includes a nonchemical process i.e., an enzymaticprocess. In this process, partitioning or fractionation preferablyproceeds as outlined hereinafter.

The stabilized rice bran is made into about a 15% to about a 35% slurry,preferably, 20-25% slurry with potable water. An enzyme, which caninclude, but is not limited to, a dextranase, a maltase, an a-amylase,and various other carbohydrate cleaving enzymes, is added to the batchconverting the starch to dextrins. The slurry is heated to about 150° F.to about 200° F. using for instance, a steam injection cooker, a heatexchanger, or other heating method. The slurry is then pumped to ahorizontal centrifuge wherein the insoluble fraction is separated. Theinsoluble fraction is collected and then dried on a belt dryer, andsubsequently ground into a powder. This powder is the stabilized ricebran insoluble fraction. The aqueous material is pumped to a drum dryerand then dried. This dried aqueous portion produces the stabilized ricebran solubilized fraction.

The enzyme-treated stabilized rice bran can be generated using the ricebran slurry as described above. The process for making an enzyme-treatedstabilized rice bran derivative can comprise admixing stabilized ricebran with an aqueous solution to form about a 15% to about a 35% aqueousrice bran slurry, preferably a 20% to about a 30% aqueous rice branslurry w/w; adding an enzyme to the aqueous rice bran slurry to convertstarch to dextrin, thereby forming an enzyme-treated slurry, and thendirectly drying the enzyme-treated slurry to form an enzyme-treatedstabilized rice bran derivative.

Preferably, after the enzyme is added to the slurry, the slurry isheated to about 150° F. to about 200° F. The slurry is then dried,wherein the drying is accomplished by a process such as belt drying,spray drying, drum drying and air-drying.

These stabilized rice bran derivatives are also available commerciallyfrom NutraStar and the RiceX™ Company of California. For the purpose ofthe invention, stabilized rice bran is available from NutraStar asStaBran™ or from RiceX™ as Stabilized Rice Bran. The soluble derivativeis available from NutraStar as RiSolubles™, non-chemically predigestedand separated from insoluble fiber, or RiceX™ Ricelin™ from the RiceX™Company. NutraStar and the RiceX™ Company are located in El DoradoHills, Calif. Rice bran oil is extracted from rice bran using standardmethods known in the art for extracting oils such as peanut oil. Theinsoluble derivative is available as RiceX™ Fiber Concentrate.

These derivatives have been shown to have more than a hundred (100)potent anti-oxidants. The major antioxidant vitamin E and its isomersknown as tocopherols (T) and tocotrienols (T₃) are collectively calledtocols. A tocol-rich substance is a mixture containing one or morecompounds selected from tocopherols (T), tocotrienols (T₃), andtocotrienol-like (T₃-like) compounds. Stabilized rice bran is thehighest natural source of vitamin E.

Antioxidants in stabilized rice bran derivatives include, but are notlimited to, γ-oryzanol, β-carotene, several known flavanoids,phytosterols, lipoic acid, and ferulic acid. Some of these compounds arepresent at a high concentration, much more than in any of the knownnatural sources.

The processing of rice bran and the nutritional composition of ricebran, as well as other aspects of the stabilized rice bran derivativesused in formulations of this invention, are further described in issuedU.S. Pat. No. 6,126,943, entitled “Method for TreatingHypercholesterolemia, Hyperlipidemia, and Atherosclerosis”, and allowedU.S. patent application Ser. No. 09/624,474, which are both incorporatedherein by reference.

Table I below sets forth the NutraStar StaBran™ Product Data Sheetcomprising stabilized Rice Bran and Germ, non-chemically treated todeactivate lipase. TABLE I NutraStar StaBran ™ Regular Product DataSheet INGREDIENTS: Stabilized Rice Bran and Germ, non-chemically treatedto deactivate Lipase and ensure total stability. GUARANTEEDSPECIFICATIONS: Protein 12-16% Soluble 2-6% Fiber Fat 18-23% Ash 7-10%Total 45-55% Moisture 4-8% Carbohydrates Total Dietary 23-35% Free Fatty<3% Fiber Acids MICROBIOLOGICAL: Total Plate Maximum 10,000 CFU/g. CountTotal Maximum 100 CFU/g. Coliform E. coli Maximum <10 CFU/g. SalmonellaNegative Yeast Maximum 100 CFU/g. Mold Maximum 100 CFU/g. PHYSICAL:Appearance Granular Solid Color Light brown/tan ¶ Flavor Nutty, ToastedBulk Density 0.47 (g/cc) ANALYTICAL DATA MACRONUTRIENTS (g/100 g)Protein (N × 6.25) 14.50 Fat 20.50 Saturated Fatty Acids 3.70 TotalCarbohydrate 51.00 Available Carbohydrate 22.00 Ash 8.00 Moisture (100degree vac.) 6.00 Crude Fiber 7.30 Total Dietary Fiber 29.00 SolubleFiber 2.00 Calories/100 g. 330.50 VITAMINS Vitamin A; Carotenoids(mcg/100 g) β-Carotene 37.00 α-Carotene 0.40 Lycopene 2.30 Lutein 63.80Zeaxanthin 18.40 Precryptoxanthin/Cryptoxanthin 7.40 Total Carotenoids129.30 Vitamin B Complex (mg/100 g) Vitamin B1 2.70 Vitamin B2 0.28Vitamin B3 46.90 Vitamin B5 3.98 Vitamin B6 3.17 Vitamin B12 (mcg/100 g)<0.500 Vitamin C(mg/100 g) <0.500 Vitamin E Complex (mg/100 g)Tocopherols (T) 12.00 Tocotrienols (T3) 13.60 Total Tocols (T + T3)25.60 Other Micronutrients (mg/100 g) Folic Acid (mcg/100 g) 26.60Biotin (mcg/100 g) 14.10 Choline 104.80 Inositol 1496.0 γ-Oryzanol245.15 Phytosterols (mg/100 g) β-Sitosterol 167.67 Stigmasterol 62.64Campesterol 96.23 Brassicasterol 14.61 Total Phytosterols 341.15MINERALS (mg/100 g) Sodium 8.00 Potassium 1573.00 Calcium 40.00Magnesium 727.00 Phosphorous 1591.00 Manganese 25.60 Iron 7.70 Copper0.27 Zinc 5.50 Chromium (ppm) <1 ppm Total Sugars(g/100 g) 8.09 (NoLactose)

Table II below sets forth NutraStar RiSolubles™ product data sheet thatcomprises stabilized Rice Bran and Germ, non-chemically predigested andseparated from insoluble fiber. TABLE II NutraStar RiSolubles ™ ProductData Sheet INGREDIENTS: Stabilized Rice Bran and Germ, non-chemicallypredigested and separated from insoluble fiber. GUARANTEEDSPECIFICATIONS: Protein  7-12% Ash 3-7% Fat 25-32% Moisture 2-7% TotalCarbohydrates 50-60% Free Fatty Acids <3% Total Dietary Fiber 0-6%MICROBIOLOGICAL: Total Plate Count Maximum 10,000 CFU/g. Total ColiformMaximum   100 CFU/g. E. coli Maximum   <10 CFU/g. Salmonella NegativeYeast Maximum   100 CFU/g. Mold Maximum   100 CFU/g. PHYSICAL:Appearance Fine Powder Color Pale Yellow Flavor Sweet, Nutty BulkDensity (G/Cc) 0.31 ANALYTICAL DATA MACRONUTRIENTS (g/100 g) Protein (N× 6.25) 7.50 Fat 26.50 Saturated Fatty Acid 4.80 Total Carbohydrate57.50 Available Carbohydrate 54.50 Ash 5.00 Moisture (100 degree vac.)3.00 Crude Fiber 4.60 Total Dietary Fiber 3.00 Soluble Fiber 3.00Calories/100 g. 486.50 VITAMINS Vitamin A; Carotenoids (mcg/100 g)β-Carotene 8.10 α-Carotene 0.00 Lycopene 0.20 Lutein 26.10 Zeaxanthin10.90 Precryptoxanthin/Cryptoxanthin 1.27 Total Carotenoids 46.57Vitamin B Complex (mg/100 g) Vitamin B1 3.60 Vitamin B2 0.46 Vitamin B376.60 Vitamin B5 5.82 Vitanin B6 5.81 Vitamin B12 (mcg/100 g) <0.500Vitamin C (mg/100 g) <0.500 Vitamin E Complex (mg/100 g) Tocopherols (T)8.00 Tocotrienols (T3) 10.00 Total Tocols (T + T3) 18.00 OtherMicronutrients (mg/100 g) Folic Acid (mcg/100 g) 36.17 Biotin (mcg/100g) 14.70 Choline 150.00 Inositol 1490.0 γ-Oryzanol 248.10 Phytosterols(mg/100 g) β-Sitosterol 211.90 Stigmasterol 68.69 Campesterol 117.32Brassicasterol 15.25 Total Phytosterols 413.16 MINERALS (mg/100 g)Sodium 15.75 Potassium 1562.00 Calcium 8.30 Magnesium 170.80 Phosphorous763.00 Manganese 3.20 Iron 1.90 Copper 0.07 Zinc 1.75 Chromium (ppm) <1ppm Total Sugars (g/100 g) 13.83 (No Lactose)

Table III below sets forth the product data sheet for Rice bran oil.TABLE III Rice Bran Oil Refined Product Data Sheet SPECIFICATIONSPhysicochemical Parameters: Appearance Clear Color (5¼″ Lovibond Red)3.5 max. Moisture (%) 0.05-0.10 Specific gravity @ 25° C. 0.910-0.920Refractive Index @ 40° C. 1.460-1.470 Iodine Value  90-105Saponification Value 180-190 Acid Value <0.5 Unsaponifiables <3% SmokePoint >213° C./415° F. Flash Point >250° C./480° F. Fatty Acid Profile(%): Myristic Acid (C14:0) 0.3-0.5 Palmitic Acid (C16:0) 14.0-20.0Stearic Acid (C18:0) 1.2-2.0 Oleic Acid (C18:1) 40-44 Linoleic (C18:2)34-40 Linolenic (C18:3) 1.0-2.0 Micronutrients: Tocopherols &Tocotrienols 500-600 ppm ANALYSIS Physicochemical Parameters: AppearanceClear Color (5¼″ Lovibond red) 3.5 max Specific gravity @ 25° C. 0.916Refractive Index @ 40° C. 1.47 Smoke Point 213° C. Iodine Value 104Saponification Value 187 Acid Value <0.5 Unsaponifiables <3% Fatty AcidProfile (%): Pahnitic Acid (C 16:0) 16.0 Stearic Acid (C18:0) 2.0 OleicAcid (C18:1) 42.0 Linoleic (C18:2) 38.0 Linolenic (C18:3) 2.0Micronutrients: Tocopherols & Tocotrienols 600 ppm

TABLE IV Antioxidants in NutraStar's Rice Bran Products Gamma Oryzanol(2200-3000 ppm) Gamma Oryzanol is not a single component. It is amixture of 20 components having different antioxidant properties.Cycloartenol trans-ferulate Cycloartenol cis-ferulate Cycloartanoltrans-ferulate Cycloartanol cis-ferulate Cycloeucalenol trans-ferulateCycloeucalenol cis-ferulate 24-Methylenecycloartanol trans-ferulate24-Methylenecycloartanol cis-ferulate 24-Methylcholesteroltrans-ferulate 24-Methylcholesterol cis-ferulate β-Sitosteroltrans-ferulate β-Sitosterol cis-ferulate β-Sitostenol trans-ferulateβ-Sitostenol cis-ferulate Stigmasterol trans-ferulate Stigmasterolcis-ferulate Stigmastenol trans-ferulate Stigmastenol cis-ferulateCampesterol trans-ferulate Campesterol cis-ferulate Tocopherols &Tocotrienols (220-320 ppm) Tocopherols and tocotrienols belong to thesame chemical group, but exist in 10 different isomeric forms havingdifferent antioxidant properties. α-Tocopherol β-Tocopherol γ-Tocopherolδ-Tocopherol α-Tocotrienol β-Tocotrienol γ-Tocotrienol δ-TocotrienolDesmethyl-tocotrienol Didesmethyl tocotrienol Polyphenols Ferulic acidα-Lipoic acid Methyl ferulate ρ-Coumaric acid ρ-Sinapic acid IsovitexinProanthocyanidins Metal Chelators Magnesium (6250-8440) Calcium(303-500) Phosphorous (14,700-17,000) Phytosterols (21 Components)(2230-4400 ppm) β-Sitosterol Campesterol Stigmasterol SitostenolΔ⁵-Avinasterol Δ⁷-Stigmastenol Sterol glucoside Acylsterol glucosideOligoglycosylsterol Monoglycosylsterol CellotetraosylsitosterolMethylsterol Dimethylsterol Gramisterol Isofucosterol ObtusifoliolBranosterol 28-Homotyphasterol 28-Homosteasteronic acids6-Deoxycastasterone β-Amyrin Carotenoids (0.9-1.6 ppm) α-Caroteneβ-Carotene Lycopene Lutein Zeaxanthine Amino Acids Tryptophan (2100)Histidine (3800) Methionine (2500) Cystein (336-448) Cystine (336-448)Arginine (10800) B Vitamins Thiamin (22-31) Riboflavin (2.5-3.5) Niacin(370-660) Pantothenic acid (36-50) Pyridoxine (29-42) Betaine Dimethylglycine Inositol (12000-18,800) Biotin (0.1-2.2) Choline (930-1150)Folic acid (0.20-0.30) Phytates (1500-1750) PolysaccharidesCycloartenol-ferulic acid glycoside Diferulic acid complex Diferulicacid-calcium complex Hemicelluloses Arabinogalactan ArabinoxylanXyloglucan Proteoglycan Glycoprotein Arabinofuranoside PhosphollpidsPhosphatidylserine PhosphatidylCholine PhosphatidylethanolamineLysophophatidylcholine Lysophosphatidylethanolamine Enzymes Glutathioneperoxidase Methionine reductase Superoxide dismutase Polyphenol oxidaseCatalase Coenzyme Q10 Aspartate amino transferase isozyme AAT-1 & AAT-2

B. Fortification Agents

Other compounds used in formulations and methods of this inventioninclude fortification agents which can include, but are not limited to,a glucosamine derivative, methylsulfonylmethane, yucca concentrate,grape seed extract, curcumin, ginger powder, boswellin, aswagandha, andhempseed oil.

Glucosamine sulfate and N-acetylglucosamine can be extracted fromcrustacean shells. Yucca concentrate is derived from the root of theyucca plant. Methylsulfonylmethane, curcumin, ginger, and boswellin,aswagandha, and hempseed oil are derived from plant sources.

C. Extracts ofActive Ingredients of Stabilized Rice Bran

In certain aspects, the active ingredients described above can also beextracted from the stabilized rice bran derivatives and usedindividually to treating inflammation, pain, lameness, and loss ofmobility.

II. ACTIVITY OF COMPOUNDS USED IN THE METHODS AND FORMULATIONS

The present invention is based on the discovery that rice branderivatives and extracts of their active ingredients are useful fortreating inflammatory reactions, pain, lameness, and loss of mobility.These components seem to act synergistically, creating an enhancedeffect not expected when one evaluates the individual compounds presentin rice bran derivatives. It is believed that the synergestic activityarises between dense phytonutrients and antioxidants present in the ricebran derivatives.

Without being bound by any particular theory, it is believed that themechanisms of action of the individual bioactive components in thestability of rice bran derivatives include, but are not limited to, thefollowing:

-   -   1. Phytosterols: Beta-sitosterol and its glycosides improve        immune function.    -   2. Gamma-oryzanol acts as an antioxidant and anti-inflammatory        agent.    -   3. Tocotrienols inhibit prostaglandin synthetase activity, which        reduces the inflammatory response by suppressing the        pro-inflammatory cytokines (IL-4) and elevating the        anti-inflammatory cytokines (IL-2).    -   4. Tocopherols act as antioxidants.    -   5. Minerals such as magnesium and other trace materials increase        absorption of calcium and help in healthy bone mineralization.    -   6. Proteoglycans, the matrix of collagen, are synthesized from        the amino acid pool and polysaccharide units from the rice bran        derivative.    -   7. Polyphenols such as ferulic acid, tocopherols and        gamma-oryzanol present in rice bran products are potent        anti-oxidants which help in joint function.    -   8. Omega-3 fatty acids in rice bran products enhance the        anti-inflammatory cytokines and inhibit pro-inflammatory        cytokines. They are also powerful COX-2 inhibitors.

The invention is further based on the discovery that the administrationof stabilized rice bran derivatives with other bioactive compounds areparticularly useful for treating inflammatory reactions, pain, lameness,and loss of mobility. Such fortified formulations are more effective,have more immediate action, and require lower dosages than currentlyexisting formulations for such conditions. In certain instances, thesecomponents act synergistically, creating an enhanced effect which isgreater than the individual compounds acting alone or additively. Thissynergy is completely unexpected when one evaluates the individualbioactive compounds present in the formulation.

Without being bound by any particular theory, it is believed that themechanisms of action of these components include, but are not limitedto, the following:

-   -   1. Yucca concentrate is rich in steroidal saponins, which        increases cortisone production. Cortisone has an        anti-inflammatory effect.    -   2. Glucosamine derivatives, such as n-acetyl glucosamine and        glucosamine sulfate, promote repair of damaged connective        tissues. Specifically, these derivatives promote cartilage        production.    -   3. Methylsulfonylmethane is a sulfur donor for the synthesis of        collagen. It maintains the synovial fluid and facilitates the        absorption of vitamin C, biotin, and panthothenic acid.    -   4. Grape seed extract is a potent antioxidant that helps prevent        free radical damage at the joints.    -   5. Curcumin and ginger powder have anti-inflammatory effects.    -   6. Boswellin is a prostaglandin synthetase inhibitor.    -   7. Aswagandha is a COX-2 inhibitor.    -   7. Hempseed oil is rich in omega-3 fatty acids, which have        antiinflammatory effects.

Although the mechanism of action of the compounds of the presentinvention discussed above are believed to be correct, it should in noway be considered as limiting the present invention. Those of skill inthe art will understand that the various embodiments of the inventionmay be practiced regardless of the model used to describe thetheoretical underpinnings of the invention.

Animal models that are widely viewed to reflect inflammatory responsesand to have predictive value in assessing the efficacy of varioustreatments for these disorders can be utilized to evaluate thetherapeutic efficacy of the compounds described herein. As described inExample 1, the effects of the compounds on joint inflammation andlameness can be assessed in horses with trauma-induced inflammation.Alternatively, improvement of inflammation and mobility can be measuredwith a collagen-induced rheumatoid arthritis mouse model (see, Enokida Met aL (2001) Bone 28(1): 87-93)

III. FORMULATION AND DOSAGES USED IN METHODS OF THIS INVENTION

The present invention also provides various formulations of stabilizedrice bran derivatives and their extracted active ingredients withfortification agents. These formulations include both neutraceuticalformulations and standard pharmaceutical compositions.

A. Nutraceuticals

The nutraceutical formulations of this invention can take a variety offorms, such as a powder, a food, a food supplement, a medical food, aliquid, a beverage, an emulsion or mixtures thereof.

To incorporate the fortified formulation of the rice bran derivativeinto the diet of a mammal various options include, but are not limitedto, simply sprinkling the formulation on another food substance (i.e.,salad, bread, cereal, etc.), being a major ingredient in a multigrainready-to-eat cereal, incorporating it into a baked product (breads,muffins, waffles, etc.), pasta, healthy dessert and snacks (athleticbar, healthy drink, etc.) and high fiber foods.

For administration to humans, the fortified formulation of rice branderivative is preferably a drink, a capsule, or a bar. Theseformulations typically comprise the solubilized fraction of stabilizedrice bran or rice bran oil. Such formulations further comprise afortification agent which promotes joint health, including, but notlimited to, glucosamine derivatives (such as n-acetyl glucosamine,glucosamine sulfate), methylsulfonylmethane, yucca concentrate, andgrape seed extract. Additives to improve flavor and consistency of theproduct are also included in the formulation. Especially preferredformulations and dosages are described in Example 2.

For administration to animals, the fortified formulation of rice branderivative is a powder or liquid. These formulations typically comprisethe solubilized fraction of rice bran or rice bran oil. Suchformulations also comprise a fortification agent, including, but notlimited to, glucosamine derivatives (such as n-acetyl glucosamine,glucosamine sulfate), methylsulfonylmethane, and yucca concentrate.Additives to improve flavor and consistency of the product are alsoincluded in the formulation. Especially preferred formulations anddosages are described in Example 2.

B. Pharmaceutical Compositions

In other preferred embodiments, formulations of this invention arepharmaceutical compositions suitable for administration via variousroutes, preferably orally or topically, and for therapeutic and/orprophylactic administration. A number of suitable formulations for usein the present invention are found in Remington's PharmaceuticalSciences (Mack Publishing Company, Philadelphia, Pa., 17th ed.,1985) andin Dermatological Formulations: Percutaneous absorption, Barry (Ed.),Marcel Dekker Inc., 1983, both incorporated herein by reference.Moreover, for a brief review of methods for drug delivery, see Langer,Science 249:1527-1533, 1990, which is also incorporated herein byreference. The pharmaceutical compositions described herein can bemanufactured in a manner that is known to those of skill in the art,i.e., by means of conventional mixing, dissolving, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes. Itwill be appreciated that the present methods and excipients are merelyexemplary and are in no way limiting.

1. Topical Administration

More particularly, these compounds can be formulated into preparationsin solid, semi-solid, or liquid forms suitable for local or topicaladministration, such as gels, water soluble (e.g., K-Y) jellies, creams,lotions, suspensions, foams, powders, slurries, ointments, solutions,oils, pastes, suppositories, sprays, emulsions, saline solutions,dimethylsulfoxide (DMSO)-based solutions. In general, carriers withhigher densities, such as K-Y jelly, are capable of providing an areawith a prolonged exposure to the active ingredients. In contrast, asolution formulation provides more immediate exposure of the activeingredient to the chosen area, although the effects generally do notlast as long.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients, which are compounds that allow increasedpenetration of, or assist in the delivery of, therapeutic moleculesacross the stratum comeum permeability barrier of the skin. There aremany of these penetration-enhancing molecules known to those trained inthe art of topical formulation. Examples of such carriers and excipientsinclude, but are not limited to, humectants (e.g., urea), glycols (e.g.,propylene glycol), alcohols (e.g., ethanol), fatty acids (e.g., oleicacid), surfactants (e.g., isopropyl myristate and sodium laurylsulfate), pyrrolidones, glycerol monolaurate, sulfoxides, terpenes(e.g., menthol), amines, amides, alkanes, alkanols, ORGELASE, water,calcium carbonate, calcium phosphate, various sugars, starches,cellulose derivatives, gelatin, and polymers such as polyethyleneglycols.

2. Oral Administration

For enteral administration the compounds useful in the methods of theinvention can be administered in either single or multiple dosages. Thecompounds may be administered in combination with pharmaceuticallyacceptable carriers in a variety of dosage forms. For example, capsules,lozenges, hard candies, powders, sprays, aqueous suspensions, elixirs,syrups, and the like may be formulated with various pharmaceuticallyacceptable inert carriers. Such carriers include solid diluents orfillers, sterile aqueous media and various non-toxic organic solvents.

Tablets may contain various excipients such as sodium citrate, calciumcarbonate and calcium phosphate, along with various disintegrants suchas starch (preferably potato or tapioca starch), alginic acid andcertain complex silicates, together with binding agents such aspolyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often very useful for tabletting purposes. Solid compositionsof a similar type may also be employed as fillers in soft andhard-filled gelatin capsules; preferred materials in this connectionwould also include lactose or milk sugar as well as high molecularweight polyethylene glycols. When aqueous suspensions and/or elixirs aredesired for oral administration, the essential active ingredient thereinmay be combined with various sweetening or flavoring agents, coloringmatter or dyes and, if so desired, emulsifying and/or suspending agents,together with such diluents as water, ethanol, propylene glycol,glycerin and various combinations thereof.

3. Dosages

Pharmaceutical compositions suitable for use in the present methodsinclude compositions wherein the active ingredients are contained in atherapeutically or prophylactically effective amount. The amount ofcompound or composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, and the manner of administration. A typical dosage forenteral administration is an amount from about 2 grams to about 100grams per day. Determination of an effective amount is well within thecapability of those skilled in the art, especially in light of thedisclosure provided supra.

As used herein, “effective amount,” or “therapeutically effectiveamount” refers to an amount of any of the compounds or formulations usedin methods of the present invention that results in treatment of themedical condition, i.e., reduction in pain, redness, inflammation,lameness, or any other symptom. Alternatively, an “effective amount” maybe determined by monitoring reduction in any detectable symptom of thecondition, such as the degree of swelling, inflammation, redness, sizeof the affected area, range of motion, and the like. In the context ofthe present invention, “prophylactically effective amount” refers to anamount of any of the present compounds that prevents the development orrelapse of a medical condition. For example, a “prophylacticallyeffective amount” is an amount that protects a subject from developingan inflammatory disorder of the joint.

For any compound or formulation used in a method of the invention, atherapeutically effective dose can be estimated initially from animalmodels (described supra), well known to those of skill in the art. Suchinformation can be used to more accurately determine useful doses inhumans. Initial dosages can also be estimated from in vitro or in vivodata.

Initial dosages can also be formulated by comparing the effectiveness ofthe compounds used in the methods of the present invention in modelassays with the effectiveness of known compounds. For instance, initialdosages can be formulated by comparing the effectiveness of thecompounds in model assays with the effectiveness of other compounds thathave shown efficacy in treating the present conditions. In this method,an initial dosage can be obtained by multiplying the ratio of effectiveconcentrations obtained in the model assay for the compounds used inmethods of the present invention and the control compound by theeffective dosage of the control compound. For example, if a compounduseful in a present method is twice as effective in a model assay as aknown compound (i.e., the EC50 of the compound is equal to one-half theEC50 of the known compound in the same assay), an initial effectivedosage of the compound would be one-half the known dosage for the knowncompound. Using these initial guidelines one having ordinary skill inthe art could readily determine an effective dosage in humans or othermammals.

Dosage amount and interval may be adjusted individually to providelevels of the active compound which are sufficient to maintaintherapeutic effect. One having skill in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

4. Combination with Other Anti-Inflammatory Agents

In pharmaceutical dosage forms, the present methods can use formulationswhere compounds are administered in the form of their pharmaceuticallyacceptable salts, or they may also be used alone or in appropriateassociation, as well as in combination with other pharmaceuticallyactive compounds. Agents of particular use in the formulations of thepresent invention include, for example, local anesthetics,counterirritants, anti-inflammatory agents, or any agent that has atherapeutic effect for inflammatory diseases or conditions.

The preferred anti-inflammatory agents include, but are not limited to,prescription and nonprescription topical and aerosol corticosteroids,non-steroidal anti-inflammatory agents including salicylates,colchicine, para-aminophenols, propionic acids, macrolideimmunosuppressives, dapsone, clobetasol, halobetasol, diflorasone,piroxicam, ketorolac, ketoprofen, indomethacin and specificcyclooxygenase inhibitors.

The preferred counterirritants include, but are not limited to,glycerol, corticosteroids and salicylates. The preferred anestheticsinclude, but are not limited to, amide caines and counterirritants withlidocaine, cocaine, bupivicaine, mepivicaine, etidocaine,chloroprocaine, proparacaine, tetracaine, benzacaine, prilocaine,benoxinate, dibucaine, dyclonine, pramoxine, menthol, resorcinol, thymoland camphor.

Any other compound that has potential efficacy in the treatment of thepresent conditions can also be used.

5. Routes of Administration

The compositions useful in the present methods can be administered to apatient using a variety of routes, such as oral, parenteral or localroutes. The present compositions are typically administered to a patientas a local application, where “local application,” or “locally applied,”refers to the administration of a composition at the local site of thedisease, whether by local injection, topical administration, or any suchmethod that results in a relatively high concentration of the compoundsused in methods of the present invention at the site of the disease. Assuch, administration of the compounds can be achieved in various ways,including by topical application of the composition to the site of thedisease or condition, i.e., direct application of a formulation to theaffected skin or mucous membrane. In addition, compositions can beformulated for injection and injected locally at the site of the diseaseor condition, e.g., local subcutaneous injection at the site of thedisease.

6. Areas for Topical Application

The compositions usefil in the present methods can be applied to anysite of any of the present conditions, including localized conditions orconditions affecting large areas of the body or even covering the entirebody, can be applied to the skin and/or to mucous membranes, and can beapplied to any affected part of the body, including the face, forehead,chin, eyes, eyelids, eyebrows, nose, skin near the nose, cheeks, ears,mouth, tongue, inside of the cheeks, gums, head, hair, scalp, neck,chest, back, lower back, armpit, skin folds of armpit, elbow, elbowfold, wrists, ankles, legs, arms, insides of wrists, insides of arms,nails, knees, area behind knees, hands, feet, palms, soles, fingers,toes, genitals, or any other affected part of the body.

7. Frequency of Administration

The compositions useful in the present methods can be administered onetime or multiple times, depending on the compound, the severity of thecondition, and the initial response of the condition to the treatment.For example, the compositions can be administered 1, 2, 4, or more timesper day, and can be administered every 1, 2, 4, 7, or more days. Suchtreatments can be administered for a limited duration, or indefinitelyuntil the condition has resolved. The compositions can be appliedlocally as a “leave on” product, meaning that the composition is appliedto the patient and allowed to remain indefinitely at the site ofapplication, or as a “wash off” product, meaning that the composition isallowed to remain at the site of application for a limited amount oftime, e.g., for a certain number of seconds, minutes, hours, etc.

It will be appreciated that the present methods of treatment can beapplied alone or in combination with other surgical or non-surgicaltreatments for these conditions.

IV. THERAPEUTIC APPLICATIONS

As discussed previously, the formulations and methods of the inventionare useful for treating inflammatory diseases or reactions. Preferably,these inflammatory diseases are disorders of the bone joint. Inespecially preferred embodiments, these methods are used to treatinflammatory diseases such as osteoarthritis, osteoporosis, rheumatoidarthritis, and soft tissue rheumatism. The term “treatment” is used torefer to a reduction of any of the symptoms associated with theseinflammatory conditions, including, but not limited to, inflammation,redness, pain, swelling, lameness, and loss of mobility.

The methods of the present invention can also be used to prevent and/ortreat inflammatory skin diseases (e.g., atopic dermatitis, eczema,contact dermatitis, allergic dermatitis), skin irritation, inflammatorypulmonary disease or reactions (e.g., asthma, allergic rhinitis, chronicobstructive pulmonary disease, and adult respiratory distress syndrome),inflammatory musculoskeletal disease or reaction (e.g., soft tissuerheumatism, exercise-induced injury, rheumatoid arthritis, psoriaticarthritis, osteoporosis and osteoarthritis), inflammatorygastrointestinal disease or urogenital reaction (e.g. enterocolitis,gastritis, Crohn's disease, interstitial cystitis, vaginitis, andulcerative colitis), autoimmune disease or reactions (e.g., inflammatorybowel disease, and psoriasis), and transplantation rejection reactions.

Furthermore, the compounds of the invention can be used to treatlameness or loss of mobility arising from the inflammatorymusculoskeletal diseases described above, as well as other conditions.In another preferred embodiment, the methods of this invention are usedto treat pain in or around a bone joint arising from the inflammatorymusculoskeletal diseases described above, as well as other conditions.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1

This example illustrates the effectiveness of fortified formulations ofstabilized rice bran derivatives for reducing lameness and jointinflammation in horses.

Clinical trials on 51 horses were conducted to determine the ability offormulations of this invention to improve lameness and reduce jointinflammation. Horses were fed with formulations of this invention andother commercially available treatments used to treat lameness. Horseswere videotaped on day 1, day 21, and day 50 of the trial. Lameness wasevaluated using the scoring system illustrated in FIG. 1. Thecorrelation between treatment with the various formulations and“lameness” is illustrated in FIG. 2. The effect of treatment with thevarious formulations on inflammation was measured by injecting the neckof the horse with carrageenan on day 21 and day 50, then observing thesize of the injection site over 7 hours (FIG. 3). Prior to initiation ofthe trial, a range- finding study was conducted to determine the amountof carageenan and route of administration necessary to induce measurableamounts of inflammation.

A range finding study was conducted to find quantifiable endpoint ofcarrageenan induced subcutaneous skin inflammation and to comparevarious commercially available oral products designed to reduceinflammation in horses and to then compare these to phenylbutazoneadministration intravenously 12 hours prior to comparison and again onehour to commencement of comparison, on day 21 and day 50 postcommencement feeding of the oral preparations. A 0.1 mL of subcutaneousinjection of 2% carrageenan in sterile water, autoclaved for sterilityprovided a reproducible challenge under the study conditions utilized. Adose of 2 grams of phenylbutazone given intravenously twice prior toeach evaluation day provided by a clinically significant reduction ininflammation for comparison.

Preparation of potential horses for the project consisted of evaluationof general health and history of clinical health and soundness. Wherepertinent, a routine lameness evaluation to verify presence of arthriticand/or lameness of long-standing duration rather that short-termlameness (bruises, soft tissue damage, etc.) that would heal with orwithout intervention. If an individual satisfied these criteria, thenroutine immunizations (tetanus toxoid, eastern-western encephalitisinfluenza, and rhino-pneumonitis) as well as cost to the owners. Oncethe trial cell was completed, containing three of five clinically lamehorses (exception trials cells “A” and “B”), then each individual wasgiven a project number for identification. Each number was then brandedinto the antero-lateral aspect of the hoof was on the left front hooffor identify verification on days #1, day#21 and day #50.

Fifty one horses (10 groups of 5 each with one extra) were fed a basaldiet consisting of alfalfa and/or grass hay, oats and stabilized ricebran as needed for consumption of the various oral products beingevaluated. Horses were not randomly selected for the trial cells, sinceat least three of five in each cell had to exhibit clinical lameness.(Cells “A” and “B” excluded from this requirement) Horses onceidentified as a part of the trial were eliminated if (1) they refused toeat the product (2) were given medication other than those included inthe project, for whatever reason that might interfere with the results.

The purpose of this study was to compare the various products, bothavailable and non-available, alone or in combination with each other andagainst phenylbutazone, the “benchmark” NSAID for tissue inflammationreductions. In addition, videos of lame horses were taken on day #1,#21, and #50 for subjective evaluation. Looking for improvement in thelameness condition presented on day #1.

A. Materials and Methods

A range finding study was conducted to find quantifiable endpoint ofcarrageenan-induced subcutaneous skin inflammation and to comparevarious nutraceutical products both commercially available and yet-to-beavailable to phenylbutazone, the “benchmark” anti-inflammatory drug inequine veterinary medicine.

In determining a quantifiable endpoint for carrageean injection 200 mLof a 2% w/v sterile preparation was made by certified compoundingpharmacist, using autoclave method for final sterilization. Six horseswere chosen for initial evaluation of the product. An area approximately8 cm×24 cm was clipped on the side of the neck mid-cervical and approx.3 cm below the mane and upon whichever side the mane flows. The area wasscrubbed with 70% alcohol-essential oil skin preparation. Immediatelysix small dots were placed using waterproof marker in the clipped areaapproximately equidistant horizontally along a line tuberculosis syringeusing a 25 gauge ⅝″ needle inserted through the rubber stopperpreviously saturated with 70% alcohol and allowed to partially dry. Inaddition, 0.2 mL. increment of 2% carrageean (w/v) was drawn into 5subsequent tuberculosis syringes and hub could thus be accounted. Animalwas then retrained and a 0.1 cc increment was then placed intradermallyunder each skin mark, starting the 25 gauge ⅝″ needle approximately3/16″ from the mark in a scrubbed area and ending with the point of theneedle under the indelible mark. No surgical gloves were used, but carewas taken to avoid post-scrub skin contamination. All six horses wereinjected intradermally as described for dogs in the canine DCV project.Four of the six horses exhibited inflammatory lumps of 12×16 mm to 18×22mm in size which enlarged in an elliptical fashion initially within 2-3hours, but did not regress measurably past the initial size even afterseveral weeks. The lumps seemed to be mildly to moderately tender withthe horses apprehensive when even slight digital pressure applied to thetest area. The initial swelling induced by the sterile waterapproximated the carrageean swellings, but regressed to near and normalwithin several hours. Since the swellings induced did not fit the trialprotocol requirement s, a second route of administration was performedon 6 different horses. In the second group of range findings studyhorses, only route of administration changed. All 0.1 cc injections wereplaced subcutaneously rather intradermally. Restraint again wasimportant as any carrageenan laced along the route of administrationupon needle withdrawal tended to induce a lingering bleb as well asinitial measurable swelling post injection at the sire of needleinduction. In some instances this was unavoidable under best ofcircumstances and restraint due to normal equine response to needlepenetration and material placement under the cutaneous later of skin. Inother instances, some micro bleeding at injection site was unavoidableand apparent. In each case, swelling was measured and recorded, sincemicro bleeding tends to resolve within several hours and certainly inless than 5 hours in each instance.

Swelling resulting from subcutaneous placement of 2% carrageenan weremostly elliptical, measurable, and transient. Sites ranged from 0mm×Omm, initially to 45 mm×60 mm after 7 hours.

After 14 days post range finding evaluation, three of the six horsesinitially used were then chosen at random, administered intravenousphenylbutazone at 1.0 gms. Per 500 pounds body weight and againchallenged with 0.1 cc of 2% carrageenan in five sites, using 0.1 ccsterile water as a control. All injections were given subcutaneously aspreviously described. Size of resulting lumps was measured immediatelyand again at hourly intervals for 7 hours. Resulting swellings wereminimal, measurable, and provided a comparison to phenylbutazone givenat the recommended dose and frequency generally used. Note however thatphenylbutzone cannot be used on a race day at racetracks sanctioned forpublic wagering. In addition AHSA (American Horse Association)sanctioned shows have specific guidelines which specify when and howmuch phenylbutazone may be administered to a competing horse. Thisinitial evaluation as well as follow-up evaluation during the testtrials suggests the benefits of phenylbutazone as it may be used tosatisfy drug-testing policy may be long gone prior to competition. Thismakes the benefits derived from oral nutraceuticals all the moreimportant and meaningful in controlling inflammation. The transitorynature of phenylbutazone benefit when used under present guidelinesplaces it more in the “band aid therapy” category than previouslyrecognized by veterinarians and horsemen alike. Nevertheless it willremain as the standard by which most responses to inflammation/lamenesstreatments are measured likely for years to come.

Fifty-one horses were selected for ten treatment groups. Horses pickedwere selected at random for age, use, breeding, weight and sex. Ageranged from 1 year to 37 years of age. Use included retired, broodmares,herd sires, western performance, hunter-jumper performance, in earlytraining, and untrained. Breeding included quarter horse and quarterhorse cross, thoroughbred cross, appaloosa, and paints. Weight variedfrom approximately 700 lbs to 1200 lbs. There were 7 intact males, 27castrated males, and 17 intact females. Horses were housed under typicalupper-scale horse facilities in two climates with 26 head innorthwestern Montana and 25 head in central Arizona, USA. Ambienttemperatures during the trial period ranged from 5 F to 106 F. All theproducts were stored at 73 F to 82 F and fed at ambient temperatures.All attempt was made to utilize horses typical of those eventuallytargeted for the product being evaluated, and under conditions typicallyseen when products comes to market. Equiflex Glucosamine Phenylbut.Carageen. Treatment Group Animals PL-100 fed. A. b. admin. c. admin. A 5NO NO NO NO YES B 5 NO NO NO YES YES C 5 NO NO COSEQUIN NO YES D 5 NO NONEXT LEVEL NO YES E 5 15 gm/day YES YES NO YES F 5 15 gm/day YES YES NOYES G 5 30 gm/day YES YES NO YES H 5 30 gm/day NO YES NO YES I 5 NO YESYES NO YES J 5 NO YES YES NO YES liquid onlyA. loading dose will be double dose for 21 days maintenance dose will besingle dose for 30 daysb. Cosequin and next level given at maximum recommended dosec. Phenylbutazone given at 1 gm/500 lbs 12 hours prior to challenge and1 gm/500 lbs 1 hour prior to challenge with carrageean Cells to containminimum of 3 clinically lame horses with the exception of cells “A” and“B”.

Fifty-one horses were selected for ten treatment groups. Horses pickedwere selected at random for age, use, breeding, weight and sex. Ageranged from 1 year to 37 years of age. Use included retired, broodmares,herd sires, western performance, hunter-jumper performance, in earlytraining, and untrained. Breeding included quarter horse and quarterhorse cross, thoroughbred cross, appaloosa, and paints. Weight variedfrom approximately 700 lbs to 1200 lbs. There were 7 intact males, 27castrated males, and 17 intact females. Horses were housed under typicalupper-scale horse facilities in two climates with 26 head innorthwestern Montana and 25 head in central Arizona, USA.

B. Purpose of the Study

1.0 A Study Conducted by White Eagle Toxicology Laboratories inDoylestown, Pa.

Under direction of DVC Biologics, L.P. of Wilmington, Del. described theeffect of egg powder on acute carrageean-induced inflammation in femalebeagle dogs. Findings indicated a significant reduction in inflammation.The purpose of this study was to compare reduction in inflammationinduced by carrageean resulting from various commercial nutraceuticalsclaiming inflammation reduction with egg powder and egg powder andEquiflex combinations using Phenylbutazone as a benchmark comparisonanti-inflammatory in the horse.

2.0 Study Schedule

Study initial date—July, 2000 Experimental termination—November, 2000

3.0 Test System:

3.1 Species: Horse

-   -   Breed: Qrt, Qrtx, Qrt-tbx, Appaloosa, Paint

3.2 Supplier: Randy Johnson Qrt Horses Diamond B Ranch Mark ArnoldGaryD. Kaufman, D. V. M Scott Davis

3.3 Age: 1 year to 37 year

3.4 Number:51

3.6 Justification for test system selection: Horses in use in typicallife-style of those anticipated as target group for commercial product.

3.7 Justification for the number of animals: This study was designed touse the fewest number of horses possible consistent with the objectiveof the study and scientific needs.

3.8 Justification for route of administration: The test product isintended to be administered as a supplement to the basal diet.

3.9 Acclimation: All animals were acclimated for at least 2 weeks priorto the study.

3.10 Selection Criteria

All the horses were evaluated for general health and lameness ifpresent. Horses were selected if they healthy, not likely to requiremedication for the next two months, and at least exhibit lameness fromchronic rather than acute inflammatory changes.

4.0 Animal Husbandry:

4.1 Housing: Horses were housed in individual stalls, runs, pastures, orcombination three.

4.2 Environmental conditions: The horses maintained according tocurrently acceptable practices of good animal husbandry.

4.3 Food: Alfalfa and/or grass hay with/without oats and supplementedwith a stabilized rice bran as needed to consume test product.

4.4 Water: Available ad libitum

4.5 Non contaminants were known to be in the feed or water that would beexpected to alter to outcome of the study.

5. Animal Identification and Randomization:

Each horse was individually identified using a hoof brand number burnedinto the anterolateral aspect of the left front hoof. Horses for eachtreatment group were not randomly assigned since three of 5 in groups“C” through “J” required a clinical lameness

6.0 Test products:

6.1 Identity: Egg powder, “Cosequin”, “Next Level”, and Equiflex.

6.2 Source of test product: Wolcott Farms, RiceX, DVC Biologics,Nutramax Laboratories & Sure Nutrition.

6.3 Storage conditions/stability: Test products were stored at roomtemperatures in a dry place protected from sunlight. Stability of theproducts under conditions of storage as stated.

6.4 Disposition of the test products: At the conclusion of this studyall unused portions are being held or disposed of as directed by studydirector.

7.0 Study Design

7.1 Range finding study: A range finding study was conducted to: (1)establish a measurable carrageenan induced inflammation on the neckunder the mane and (2) to verify that phenylbutazone given in anaccepted manner would significantly reduce inflammation caused bycarrageenan to use as a comparison.

During attempts to establish a measurable carrageenan inducedinflammation using route of administration suggested by DCV Biologics.Multiple attempts were unsuccessful. Inflammation did not show apatterned increase in size of bleb and transitory swelling suggested.Rather it result in a small painful lump that frequently did notincrease nor dissipate in any set pattern, but rather lingered for weeksand sometimes months as a painful, obvious swelling.

A second model was evaluated using subcutaneous route of administrationwith all other parameters being identical. A transitory lump wasproduced with almost all evidence being gone in 24-40 hours. Inaddition, the swellings were quite measurable over 7 hour period,increasing and in some instances starting to decrease within that timeframe.

The swellings were for the most part elliptical or at least notperfectly round. Inflammation in horse skin id often manifest in a rash”appearance rather than round lumps, so the shape of swelling was of noreal surprise. Measurements were taken using basically height x width ofthe swellings to establish a more representative number to describe thereaction to the carrageenan-induced inflammation. It should be notedthat swelling is a 3 dimensional phenomenon as each lump had thicknessas well as height and width. In some horses, measurements went from asignificant width x height to 0×0. In these cases the swelling meltedaway first in the middle of the lump and eventually dissipated at theborders to the extent no readings were possible. This again is notsurprising, since skin swellings in horses often vanish in this manner.Strictly reading the numbers in these cases may be quite confusing. Inthis trial, swellings were simply identified, measured and numbered andwhen identification was no longer possible, measure was called 0×0.

7.2 Efficacy study of the tests products: Al tests products weremeasured using and electronic gram scale for solids and graduated dosesyringe for liquids. Each product was measured and a daily amount wasmeasured was placed in an individual plastic container and heat-sealedusing a Food saver Bag Vac Sealer. All liquids were placed in a bagwithin a bag and heat-sealed. Each bag was then labeled with the horse'sname and identification number. Each product was mixed as requestedunder protocol treatment chart. Only the product to be given from day 1to day 21 was issued to complete the trial. Treatment group “J” did notreceive daily liquid Equiflex after day 21 challenge simply becauseproduct did not arrive form Rice-X as requested in a timely fashion.Upon arrival, product was fed as a loading dose for 6 days to compensateand ultimately allow for total amount of product in the horses by day51. This variance was discussed with and approved by Win of WolcottFarms as the logical correction to make. Cosequin was fed twice dailythree level scoops each time. This was continued throughout the entire51 days rather than drop to maintenance dose, since the label suggeststhat the horse should be watched for “movement and attitude” and thatmaintenance or transitional dosage of a lesser amount “may be increasedat any time if needed”. Rather than get it an issue, since comparisonsget into an issue, since comparisons were being drawn, Cosequin was fedthroughout at maximum recommended dose. Next level was fed at maximumdose of 2 oz throughout the first 21 days rather than the first 10 days,and then dropping to 1 oz per day as the label suggests. Again this wasto ensure maximum results. Egg biologics was fed at 15 gm/day and 30gm/day. Each was doubled during the first 21 days. When used incombination with equiflex/glucosamine both egg biologics andequiflex/glucosamine were doubled in amount. Equiflex was fed at a doseof 2.75 oz as a standard dose and a 2.72 oz when fed with egg biologics.Liquid Equiflex was fed 4.25 oz/day maintenance, 8.5 oz/day loadingdose.

8.0 Antemortem Observations

8.1 Local observations: Each animal was observed at least twice a dayduring feeding time and any changes noted. #59 Bowie was eliminated fromthe trials after day #5 for refusal to eat the loading dose of 60gms/day egg biologics with 5.44 oz day Equiflex powder. #7 Dolly wasgiven intravenous flunixamine on day 20 and scratched from the day 21evaluation, but included in the day 50 evaluation. #1 Copper waseliminated from the trial after carrageenan included lumps persisted onhis neck for more than two weeks post-injection. #18 Mercedes waseliminated on day 23. He required continuous medication for gastriculcers, which could potentially alter trial results.

8.2 Data collection: All measurements were taken by the study directorand recorded by support personnel. Measurements were made hourly for 7hours with most being on the hour with few varying several minutes,depending on the difficulty experienced in restrainingfractious/dangerous animals. TITLE: Evaluation Of The Anti-InflammatoryEfficacy Of PL-100 (a whole egg product, ex DCV), ‘Arthreflex’, ‘NextLevel’ and ‘Cosequin’ on Horses versus ‘Bute’ PURPOSE: Assess thestatistical and clinical efficacy of two test products (PL-100 in aninert carrier base and in combination with ‘Arthreflex’) versus threemarket controls in i) an equine model of inflammation, and ii)clinically lame horses. ANIMALS: SPECIES: Equine STRAIN/BREED: Variety -To be documented SEX: To be documented AGE: 3 mo-25 years SOURCE: to bedocumented Method of IDENTIFICATION: Total NUMBER: 50 Treatments: 10Number per 5 horses per TEST GROUP: treatment Number of 1 REPLICATES :Type and Stabled horses description of HOUSING: Husbandry, All horsesare vaccinations, immunized general management: according to localstandard prior to starting RATION/ BASAL DIETS DIET: Source: Alfalfaroughage diet with additional grain Nutrient Protein 14%-Specifications: 20% Basal diets are based upon NRC Formula:Nutritionally adequate and on that is commonly utilized for feeding thetype of horse used in the trial. Medication: None EXPERIMENTAL Total #50 Horses: DESIGN: Total # random Males/Females: Total # 10 TreatmentGroups: Total Duration of 50 days Treatment: Method of To be documentedRandomization: Blinding: None Statistical Student's t-test, Analysis:ANOVA & Bonferroni's Multiple Comparison post-test.

TREATMENT GROUPS: Treatment #'s PL-100 Phenylbutazone Carrageenan GroupAnimals Fed^(a) Arthreflex Glucosamine^(b) Administered^(c) AdministeredA 5 No No No No Yes B 5 No No No Yes Yes C* 5 No No Cosequin No Yes D* 5No No Next Level No Yes E* 5 15 gm/day Yes Yes No Yes F* 5 15 gm/day NoYes No Yes G* 5 30 gm/day Yes Yes No Yes H* 5 30 gm/day No Yes No Yes I*5 No Yes Yes No Yes J* 5 No Equiflex Yes No Yes Liq^(a)Loading dose will be bid for 21 days Maintenance dose will be sidfor 30 days^(b)1800 mg twice a day^(c)to be documented*Cells to contain minimum of 3 Clinically Lame HorsesExperimental Design:

Rangefinding Study: A rangefinding study was conducted to establish aquantifiable end point of carrageenan induced inflammation and todetermine the dose of phenylbutazone which will reduce the inflammationby approximately 50%. Based on these results, the procedure forchallenge will be as follows:

An area on the body will be clipped and six small lines will be drawn onthe neck with marker. Five (5) 0.1 mL intradermal injections of 2%carrageenan in sterile water will be made in the clipped area and asingle injection of water will serve as a control. The diameter of theinjection sites will be measured with calipers and recorded immediatelyafter injection and hourly there after for 7 hours following injection.5 test sites and 1 control (water) per horse, therefore 25 test sitesper test cell+5 controls

All PL-100/Glucosamine treatments will be mixed by DuCoa prior toapplication and shipped to the test facility. Retention samples ofPL-100 will be retained for assay by DCV. Retention samples for allproducts will be retained for assay by WFY. Prior to initialization ofthe trial, horses will be selected the basis of comparable size andgeneral good health. Animals will be grouped by treatment as describedin the treatment table above. Horses given PL-100 will receive a loadingdose (defined as the maintenance dose given twice a day) for 21 days.Horses on market control products will follow manufacturer'sinstructions. On day 21 all horses will be challenged with carrageenanand evaluated for inflammatory response. Ideally, the same person shoulddo the intradermal injections and the same person should do themeasuring of the inflammatory response. Starting on day 22, the horsesreceiving PL-100 will be given PL-100 once a day as a maintenance dose.All horses will then be challenged with carrageenan on day 50

Clinically Lame Horses—Observational Methods

Prior to the start of the trial each clinically lame horse will beexercised recorded by videotape. The video evidence should clearlyillustrate the animals' stride, extension and general mobility. The samerecord of the animals' physical exhibit should also be kept at day 21and day 50 of the trial. The performance difference should be evaluatedacross these three intervals by the same evaluator. The evaluators'comments and observations will be part of the trial record. The sameevaluator should be used for each of the clinically lame horses.SCHEDULE: Date Treatments August (day 0) House horses Start trialfeeding Size test August (day 21) Challenge horses September (day 50)Measure results

DATA MEASUREMENTS & RECORDS: Data Collected or Measurement Record dailyobservations for general health and disease status throughout trialperiod Record reasons for any removal of animals & specific exclusioncriteria met Record disposition of all animals and materials and attachreceiptsMETHOD OF Group mean values (+Std. Devns.). Pre- vs Post-comparisonswithin DATA ANALYSIS each treatment group conduct using paired Student'st-test. Between & STATISTICS: group comparisons using one-way ANOVA.Where significant differences (p≦0.05) are seen, use Bonferroni'sMultiple Comparison post-test to determine which means are significantlydifferent from each other.

Example 2

This example sets forth preferred formulations of this invention. HumanFormulations NutraFlex ™ (For Humans) Serving Dose 10 grams/dayRiSoluble ™   79% ± 5 Yucca   10% ± 1 Glucosamine Sulfate   5% ± 1N-Acetylglucosamine    3% ± 0.3 Methylsulfonylmethane (MSM)    2% ± 0.3Grape seed extract    1% ± 0.3 NutraFlex: Drink NAG    3% ± 0.5 MSM   2% ± 0.3 Yucca   10% ± 1 Glucosamine Sulphate   5% ± 1 Grape seed ext   1% ± 0.1 Boswellin    1% ± 0.1 Solubles   78% ± 5 Dose  10.0 g/dayNutraFlex: Capsules RiSolubles 28.0% ± 3 N-Acetyl Glucosamine 11.11% ±1  Glucosamine sulphate 16.67% ± 2  Methyl Sulfonyl Methane 11.11% ± 2 Grape seed extract 5.58% ± 1 Yucca Concentrate (70%) 5.58% ± 1 Hemp seedoil 5.58% ± 1 Boswellin 5.58% ± 1 Ginger powder 5.58% ± 1 Curcumin (95%)5.58% ± 1 Total 1.80 g for 4 capsules two with breakfast and two withdinner NutraFlex: Bar Solubles 48.39% ± 3  N-Acetyl Glucosamine 8.06% ±2 Glucosamine sulphate 8.06% ± 2 Methylsulfonylmethane 16.13% ± 2  Yuccaconcentrate   3.23% ± 0.5 Grape seed extract   3.23% ± 0.5 Aswagandha  8.06% ± 0.7 Ginger powder   3.23% ± 0.5 Curcumin   1.61% ± 0.3

Formulations for Horses EquiFlex ™ (For Horses) Serving size 2.75 oz/day(78 grams/day) Stabilized Rice Bran 88.47% ± 5   Yucca 5.13% ± 1  Glucosamine Sulfate 3.84% ± 0.5 N-Acetylglucosamine 1.28% ± 0.3Methylsulfonylmethane (MSM) 1.28% ± 0.3 Total 100% EquiFlex-LiquidFormulation Rice Bran Oil 30-60% Glucosamine Sulphate 2.62% ± 0.5N-Acetylglucosamine 0.87% ± 0.2 Methylsulfonylmethane 0.87% ± 0.2 YuccaConcentrate (70%)  3.5% ± 0.3 Water 30-60% Gum (Emulsifier)  0.2% ± 0.05Sodium benzoate(Stabilizer)  0.15% ± 0.03 Citric acid (Preservative) 0.6% ± 0.1 Apple flavor  0.25% ± 0.05 Dose 155 g/dayTopical Formulations

Arthritis Cream

-   -   Distilled Water    -   EPA    -   Toco    -   Emu Oil    -   Stabilized Oxygen    -   Orange Oil    -   Silica    -   Colloidal Silver    -   Lemon Oil    -   Poly Base    -   Yucca    -   Glucosamine sulfate    -   Methylsulfonylmethane

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification in their entirety for all purposes. It is understood thatthe examples and embodiments described herein are for illustrativepurposes only and that various modifications or changes in light thereofwill be suggested to persons skilled in the art and are to be includedwithin the spirit and purview of this application and scope of theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference in their entirety forall purposes.

1-47. (canceled)
 48. A method for treating a disorder of the immunesystem in a mammal, said method comprising: administering a fortifiedformulation comprising a stabilized rice bran derivative and at leastone fortification agent, thereby treating said disorder in said mammal.49. The method of claim 48, wherein said stabilized rice bran derivativeis a member selected from the group consisting of rice bran oil,enzyme-treated stabilized rice bran, a solubilized fraction, and amixture thereof.
 50. The method of claim 48, wherein said fortificationagent is a member selected from the group consisting of a glucosaminederivative, methylsulfonylmethane, yucca concentrate, and grape seedextract.
 51. A method for treating a disorder of the immune system in amammal, said method comprising: administering a fortified formulationcomprising a stabilized rice bran derivative and at least onefortification agent, wherein said fortification agent is a memberselected from the group consisting of a glucosamine derivative,methylsulfonylmethane, yucca concentrate, and grape seed extract,wherein said stabilized rice bran derivative is a member selected fromthe group consisting of rice bran oil, enzyme-treated stabilized ricebran wherein the enzyme is carbohydrate cleaving enzyme, a stabilizedrice bran solubilized fraction, and a mixture thereof, thereby treatingsaid immune system disorder in said mammal.
 52. The method of claim 48or 51, wherein said administering comprises ingestion of said fortifiedformulation.
 53. The method of claim 48 or 51, wherein said mammal is amember selected from the group consisting of a human, a horse, a cat,and a dog.
 54. The method of claim 53, wherein said disorder of theimmune system is an autoimmune disease.
 55. The method of claim 54,wherein said autoimmune disease is selected from the group consisting ofmultiple sclerosis, type-1 diabetes mellitus, Graves' disease, systemiclupus erythematosus, scleroderma, psoriasis, intestinal bowel diseaseand celiac disease/gluten sensitivity.
 56. The method of claim 55,wherein said mammal is a human.
 57. The method of claim 55, wherein saiddisorder of the immune system is intestinal bowel disease, and whereinsaid formulation is ingested in the amount of about 2 grams to about 100grams per day total.
 58. The method of claim 48 or 51, wherein saidformulation is a member selected from the group consisting of a cream, acapsule, a bar, a powder, a food, a food supplement, a medical food, aliquid, a beverage, an emulsion or mixture thereof.